ABSTRACT ? Characterization Unit The Breast Pre-Cancer Atlas (B-PCA) will elucidate the imaging, molecular, and microenvironment differences in DCIS that influence progression to invasive cancer. The objective of the Characterization Unit is to test and implement a multi layered experimental architecture on the biospecimens obtained in the Biospecimen Unit. The goal will be to study pre-cancers at the organ, duct and single cell resolution levels. The Characterization Unit will use approaches that are multidimensional and multiparametric while addressing the central conceptual themes of cancer progression, ecology and evolutionary biology, and breast cancer molecular subtypes. The Characterization Unit is comprised of 3 primary sites (DFCI, Duke and Stanford) which will each manage assay implementation. The assays performed include RNA-seq (both single duct and single cell), DNA-seq (WGS, WES, and in situ mutation analysis), multiplex immunohistochemistry, multiplex FISH, H&E morphometric analysis, methylation analysis, immune analysis, and radiographic imaging studies. All characterization assays used for B-PCA Atlas construction are not only robust in very limited sample size, but they can often be performed using material obtained from a single DCIS duct from one thin section. Registration of these multiple data types derived from in situ assays on thin sections will provide a platform for complex multi-dimensional data representation. Integration between the Biospecimen and Data Analysis Units of the B-PCA will be promoted by the shared data infrastructure, including access to metadata to facilitate sharing of samples and data. The Aims of the Characterization Unit are: 1) Test and standardize technologies on retrospective cohorts which will integrate genomic, transcriptomic, proteomic, and imaging methods at organ, ductal and single cell scales, 2) Apply promising technologies from Aim 1 to prospective cohorts, and 3) Deliver integrated data to the Data Analysis Unit.